At the core of µParaflo® technology, there are three components (Figure 1): a novel solution photochemistry that uses photogenerated reagent precursor (PGR-P) and conventional compounds for synthesis, digital photolithography that uses a graphic projector to direct photoreactions on chip, and microfluidic devices that provides picoliter scale reaction chambers (picoarray reactor) to allow parallel chemical and biochemical reactions1.
As shown in Figure 1, a PGR-P may be a photogenerated acid precursor (PGA-P), a compound that decomposes to form an acid upon light irradiation. The acid generated can in turn affect a reaction which normally occurs under acid conditions.
The PGA chemistry employed by this technology brought in a major advantage that made the array synthesis of a variety of molecules, such as not only oligonucleotides but also peptides and peptidomimetics, practically possible. This is because unlike another method of light-directed synthesis2,3, which uses photolabile protecting groups for amino acids (Figure 2), the PGA chemistry uses commonly used chemical supplies.
Comparatively, the photolabile group protected amino acids are impractical to use since they would be difficult and costly to make and their reaction efficiency is less than conventional chemistry which have been extensively studied and well-developed for more than three decades. Indeed, the photolabile protected amino acids have not been shown in all 20 natural amino acids and peptide chips thus synthesized were only shown in concept proven publications.2,3
Figure 3 illustrates the synthesis process of peptide arrays using the µParaflo™ technology. The key step is digital light controlled irradiation at pre-selected sites. Light irradiation causes acid (H+) to form within the sites for the next step coupling.
The overall synthesis from sequence text file to final image results is digitally encoded and can be performed on a wafer-scale in a similar way to fabrication of computer chips.
There are a number of advantages in this method:
- The synthesis method is suitable for different classes of molecules, such as oligonucleotides, peptides, or their analogs
- The synthesis can be performed in a regular research laboratory
- The synthesis efficiency can be optimized to be comparable with conventional synthesis
- It offers flexibility in the sequences to be synthesized – editing the changes in sequence text files to create different chips easily
- The overall consumption of chemicals (in synthesis) and samples (in assays) is significantly reduced to the level of sub-nanoliter to picoliter per assay
- The microfluidic reaction chambers are particularly suited for parallel biochemical reactions
- The liquid delivery to the microchip is simple (parallel flow through the inlet and outlet holes), can be automated, and there is little chance of ambient contamination
- The spot density of the chip can be ten-fold higher than that of spotted chip
Technology and Application Articles
- Gao, X., Yu, P. Y., LeProust, E., Sonigo, L., Pellois, J. P., and Zhang, H. (1998) Oligonucleotide synthesis using solution photogenerated acids. J. Am. Chem. Soc. 120, 12698-12699.
- LeProust, E., Pellois, J. P, Yu, P., Zhang, H., Srivannavit, O., Gulari, E., Zhou, X., and Gao, X. (2000) Combinatorial screening method for synthesis optimization on a digital light-controlled microarray platform. J. Comb. Chem. 2, 349-354.
- Pellois, J. P, Wang, W. and Gao, X. (2000) Peptide synthesis based on t-Boc chemistry and solution photogenerated acids. J. Comb. Chem. 2, 355-360.
- Leproust, E., Zhang, H., Yu, P., Zhou, X., Gao, X. (2001) Characterization of oligodeoxyribonucleotide synthesis on glass plates. Nucleic Acids Res. 29, 2171-2180.Text Box: TECHNICAL BULLETINGao, X., LeProust, E., Zhang, H., Srivannavit, O., Gulari, E., Yu, P., Nishiguchi, C., Xiang, Q., Zhou, X. (2001) Flexible DNA chip synthesis gated by deprotection using solution photogenerated acids. Nucleic Acids Res. 29, 4744-4750
- Pellois, J. P., Zhou, X., Srivannavit, O., Zhou, T., Erdogan, G., and Gao, X. (2002) Individually addressable parallel peptide synthesis on microchips. Naure. Biotechnol. 20, 922-926.
- Rouillard, J. M., Lee, W. Truan, G., Gao, X., Zhou, X. and Gulari, E. (2004) Gene2Oligo: Oligonucleotide design for in vitro gene synthesis. Nucleic Acids Res. 32, W176-180.
- Zhou, X., Cai, S., Hong, A., Yu, P., Sheng, N., Srivannavit, O., Yong, Q., Muranjan, S., Rouillard, J. M., Xia, Y., Zhang, X., Xiang, Q., Ganesh, R., Zhu, Q., Makejko, A., Gulari, E., and Gao, X. (2004) Microfluidic picoarray synthesis of oligodeoxynucleotides and simultaneously assembling of multiple DNA sequences. Nucleic Acids Res. 32, 5409-5417.
- Srivannavit, O., Gulari, M., Gulari, E., LeProust, E., Pellois, J. P., Gao, X., Zhou, X. (2004) Design and fabrication of microwell array chips for a solution-based, photogenerated acid-catalyzed parallel oligonuclotide DNA synthesis. Sensors and Actuators A. 116, 150-160.
- Tian, J., Gong, H., Sheng, N., Zhou, X., Gulari, E., Gao, X., and Church, G. (2004) Accurate multiplex gene synthesis from programmable DNA chips. Nature 432, 1050-1054.
- Gulari, E., Gao, X., and Zhou, X. (2003) “Light directed massively parallel on-chip synthesis of peptide arrays with t-Boc chemistry”. Proteomics 3, 2135–2141.
- Gao, X. (2004) “In situ parallel synthesis of addressable peptide microarrays” in Proceedings of the 7th China Peptide Symposium. Peptides. Biology and Chemistry. Eds. Du, Y-C., Zhang, Y. S., and Tam, J. P. Shanghai Scientific & Technology Publishers. pp. 29-33.
- Gao, X., Pellois, J. P., Kim, K., Na, Y. , Gulari, E., and Zhou, X. (2004) “High density peptide microarrays. In situ synthesis and applications”. Molecular Diversity. 8, 177-187.
- Gao, X., Gulari, E., and Zhou, X. (2004) “In situ synthesis of oligonucleotide microarrays”. Biopolymers. 73, 579-596.